Most of our PET-CT studies to date have used 18F-2-fluoro-2-deoxyglucose (FDG) to image the metabolism of the eukaryotic cells in TB lesions but we are also making attempts to identify the location, abundance and metabolic state of the bacteria in lesions. In an effort to identify small molecules that could be used to specifically label MTb in vivo, we capitalized on the unusually broad substrate tolerance of the MTb antigen 85 enzymes, which transfer mycolates onto structurally diverse sugars to form part of MTbs cell wall. Antigen-85 enzymes are expressed on the exterior of MTbs cell wall and incorporate exogenous trehalose (a nonmammalian disaccharide consisting of a two 1-1 &#945;,&#945; -linked glucose monomers) as either the mono- or dimycolate, even tolerating trehalose molecules containing bulky modifications. We have used this system to incorporate 18F trehalose into bacteria in the lesions of infected rabbits and 18F activity has been detected in lesions of infected rabbits by PET-CT imaging. A series of different positions and methods for attaching the 18F to the sugar are being explored to see which is most efficiently incorporated. Using trehalose should afford an improvement over the currently used 18F-FDG, as glucose is used by mammalian cells as well as bacterial, causing noise in the scans due to increased metabolism or inflammation in the host. Use of trehalose is unique to bacteria; it is not absorbed by mammalian cells, which should limit noise in PET scans. The Davis group has designed three trehalose analogs incorporating fluorine at the 2, epi-4, or 6-position of trehalose for use as PET radiotracers. The 2-fluorotrehalose (FDT) synthesis is a biomimetic process, inpired by bacterial synthesis of trehalose from glucose. Chemoenzymatic synthesis of FDT occurs as a one-pot cascade reaction in which hexokinase transfers a phosphate from adenosine triphosphate (ATP) to FDG (normally glucose in bacterial trehalose synthesis). OtsA then transfers the glucose from the donor UDP-glucose to the acceptor phosphorylated FDG. Dephosphorylation to give the desired product is effected by OtsB. The entire one-pot process is complete in 45 min. The advantage here is that a relatively technically facile manipulation would convert a commercially available radiotracer to a TB-specific one. We have acquired some preliminary PET-CT scan data in rabbits using the FDT probe as well, one healthy, three infected with HN878 MTb. In the first infected animal, four lesions were present. Two were not PET-active, and two were, although all four had similar amounts of colony forming units (CFU). Upon necropsy, the two PET-inactive lesions were extremely rigid and thick-walled, implying that uptake is related more to accessibility (i.e. vasculature) than amount of bacteria present. Intriguingly, the second two rabbits showed more complex lesions which clearly displayed differential labeling between the 18F-FDG and FDT. Metabolism data was inconclusive but suggested some metabolism (as much as 20%) of the probe from FDT back info FDG. It is assumed this happens via trehalase natively expressed by the rabbits. We then evaluated FDT in Mtb-infected marmosets, as marmosets should express lower levels of trehalase and are a more physiologically relevant model to human disease. The marmosets showed no metabolism of the FDT back to FDG. The FDT continued to show differential labeling compared to FDG. We were able to see the differential labeling both qualitatively and quantitatively. Areas in the top 25% of FDG uptake show disproportionately lower FDT uptake, and the areas in the top 25% of FDT uptake show disproportionately lower FDG uptake. The intriguing finding suggestes that bacterial are actively replicating in areas of lower inflammation than they are in areas with high levels of inflammation. It is premature to draw correlations between bacterial load and tracer uptake; however, it appears that the FDT may be a more reliable predictor of treatment success or failure. In one marmoset, we treated with the first-line regimen of izoniazid, pyrazinamide, rifampicin, and ethambutol for five weeks, with dosing five days a week. The animal showed very low disease burden upon necropsy as judged by CFU. The FDT PET-CT scans reflected this low burden, showing a clear reduction in uptake compared to pre-treatment scans. The FDG did not show a significant decrease in uptake and in some lesions displayed increase uptake. This is an extremely promising sign that the FDT will be able to give an earlier indication of treatment success or failure as compared to FDG. We have performed biodistribution studies by analyzing tissue samples from relevant organs in two marmosets, which showed little uptake in uninfected tissue other than kidneys, the route for clearance of the radiotracer. We have also successfully performed a biodistribution study via PET-CT in a macaque and have two additional scans scheduled for Fall 2015. These scans will give dosimetry data in preparation for moving the tracer forward for human use. We have optimized protein immobilization onto solid support. Each enzyme shows best performance on different resins. All perform acceptably using cyanogen-bromide linking onto agarose, but all three perform better on Enzyme Carrier Resins, with linker type optimized for each protein. Under optimized reaction conditions, these immobilized proteins provide good conversion which is complete in 1.5 h. With these immobilized enzymes, radiopharmacists could pass through commercial FDG and a solution of ATP and UDP-Glucose through the cartridge to produce the desired radiotracer with no specialized equipment or special training. The procedure would be operationally simple and allow TB-specific imaging to monitor course of treatment. Ideally, we would be able to rapidly assay treatment success or failure in a manner that relies only on abundance and metabolic state of the bacteria.